Microbiology 4102 Lab Practical #1 Diagram

Body Tube

Houses the lens system that magnifies the specimen.

Oculars/Eye Pieces

What you view through.

Nose-Piece

Revolves and contains the objectives.

Condensor Lens (Abbe)

A two-lens system that collects and concentrates light from the illuminator and directs it to the iris diaphragm.

Objective Lenses

Magnification ranges from 10x to 40x; found on the nose-piece.

Coarse Adjustment

Brings the specimen into general focus.

Fine Adjustment

Moves the stage slightly to sharpen the image.

Stage

Supports the slide being viewed.

Illuminator

The light source in the base of the microscope.

Iris Diaphragm (Below Stage)

Regulates the amount of light entering the lens system.

What is the total magnification for a 4X objective lens?

40X (4X objective x 10X ocular lenses).

What is the total magnification for a 10X objective lens?

100X (10X objective x 10X ocular lenses).

What is the total magnification for a 40X objective lens?

400X (40X objective x 10X ocular lenses).

What is the total magnification for a 100X objective lens?

1000X (100X objective x 10X ocular lenses).

What steps should you take when your slide is blurry under oil immersion?

Use the lens cleaning solution and a lens cleaning paper to wipe away any oil and clean the lens; then reapply immersion oil and refocus using the fine adjustment.

Describe how to use the par-focal nature of your microscope when observing a sample at 1000X total magnification.

There only needs to be a small adjustment made using the fine adjustment when moving from the 40X objective lens to oil immersion under 100X objective lens.

Describe how you take advantage of the par-central feature of your microscope.

When changing objective lenses, the area of the specimen that is already in the field-of-view will remain in the middle of the field.

What is the purpose of adding oil when using the 100X objective lens?

It diminishes the loss of refracted light (has same refractive index as glass) and improves resolution at 1000X magnification.

How do you properly carry a microscope?

One hand holding it by the handle, one hand supporting the base.

What kind of paper do you use to clean oil off a microscope lens?

Lens paper.

How do you properly store a microscope?

Microscopes in their correct number spot in the cabinet, oculars facing the back, covering placed over.

At what settings should the microscope be put away at?

4X objective lens, switched off, stage all the way lowered.

When looking at your wet mount under the microscope what did you notice about the difference in size between the bacterial cells and the cheek cells? Blood cells vs. bacterial cells?

Cheek cells are much larger than bacterial cells; blood cells are larger than bacterial cells.

What is the purpose of adding the hair strand to the wet mount?

The hair is used to establish a plane of focus.

What does a coccus cell look like?

Round; singular arrangement.

What is a diplococci arrangement?

Two round cells.

What are clusters in an arrangement?

Random grape-like clusters of cells.

What are pairs in an arrangement?

Two cells together.

What are rods?

Bacillus (rod-shaped bacteria).

What are chains in an arrangement?

Long strips of cells.

What are tetrads in an arrangement?

Four cells together.

What tool should be used to inoculate a plate?

Inoculating loop.

What tool should be used to inoculate an agar deep?

Inoculating needle.

Describe the steps you would take to ensure aseptic conditions when transferring microorganisms from one culture to another.

1. Make sure the incinerator is properly heated before use.

2. When transferring cultures, ensure the loop/needle is sterilized prior to collecting the culture and immediately after it has been transferred.

3. Make sure loop has been properly cooled before collecting cultures.

4. Properly label all cultures, NOT on lid (name, date, section, sample).

5. Minimize exposing cultures to the open air.

6. Dispose of lab waste in designated locations.

What does subculturing mean?

Transferring microorganisms from one media type to another.

Is this aseptic technique correct?

Use the loop immediately after taking it from the incinerator.

No, the loop needs to be properly cooled to prevent the generation of aerosols and to not kill the cultures.

Is this aseptic technique correct?

All cultured must be properly labeled with name, date, section, and culture.

Yes.

Is this aseptic technique correct?

Keep culture tubes open even when not in use.

No, cultures should be closed unless in use to prevent contamination from the air or your breath.

What micropipette should you use to transfer 150 microliters?

P200.

What micropipette should you use to transfer 20 microliters?

P20.

What micropipette should you use to transfer 157 microliters?

P200.

What micropipette should you use to transfer 634 microliters?

P1000.

If a P20 micropipette displays the numbers "136" in the window, what volume is the pipette set to transfer?

13.6 microliters.

If a P200 micropipette displays the numbers "198" in the window, what volume is the pipette set to transfer?

198 microliters.

If a P1000 micropipette displays the numbers "095" in the window, what volume is the pipette set to transfer?

950 microliters.

What would 386 microliters look like on a micropipette window and what micropipette should be used?

Window = "038"; P1000.

What would 170 microliters look like on a micropipette window and what micropipette should be used?

Window = "170"; P200.

What would 15 microliters look like on a micropipette window and what micropipette should be used?

Window = "150"; P20.

What is the normal media that we typically use in plate form?

Tryptic Soy Agar (TSA) plate.

What is the media that we use that is in a large glass test tube?

BHA agar deep.

What is the media that we use in a normal test tube?

Nutrient broth.

What is the media that we use that is slanted in a test tube?

TSA agar slant.

What do you write on a plate to label it?

Name, date, section, sample.

How do you properly inoculate an agar slant from an agar plate?

Sterilize inoculating loop, pick up a colony, streak in swiping motions on top of slant.

How do you properly inoculate an agar plate from an agar slant?

Sterilize inoculating loop, streak surface of agar slant, streak surface of plate in swiping motions.

How do you properly inoculate broth from an agar plate?

Sterilize inoculating loop, pick up a colony, dislodge it and mix into broth.

How do you properly inoculate an agar plate from broth?

Sterilize inoculating loop (or use pipettor), mix solution, streak surface of agar plate (or use pipettor to place on surface and spread).

How do you properly inoculate an agar deep from an agar plate?

Sterilize needle, pick up a colony, stab into agar deep.

How do you properly inoculate a plate from an agar deep?

Sterilize need, stab into agar to pick up bacteria, streak on plate.

What part of the microscope would need to be adjusted to increase or decrease the contrast during a wet mount? Where on the microscope is this part located?

The iris diaphragm would need to be adjusted to almost closed to create contrast necessary to observe cells; located under the stage.

Why would a coverslip be used to observe bacteria in a wet mount slide?

A coverslip is used to sandwich the liquid sample so that the objectives are not contaminated and to reduce the thickness of the sample.

What is the ranking of the size of E. coli, S. cerevisiae, and cheek cells?

E. coli < S. cerevisiae < cheek cells.

What is a pure culture?

A culture that only contains a single bacterial species that originated from a single cell.

Based on what characteristics can one ensure that they have obtained a pure culture on their plate?

They look homogenous and exhibit consistent biochemical characteristics that facilitate their identification.

Why would there be no growth in the 3rd quadrant on a streaked plate?

The plate was improperly streaked, did not overlap into the bacteria.

What is the method that we use to isolate organisms on plates?

T-streaking.

What may you observe is the loop was not cooled down enough when collecting bacteria to begin your isolation?

No bacterial growth because it was killed by the hot inoculating loop.

What could happen if you overheat the slide during the heat-fixing process?

Gram-positive cells stain light purple or pink.

What could happen if you underheat the slide during the heat-fixing process?

Nothing would be visible on the slide.

Why is it important to heat-fix the slide before applying certain stains, such as the gram-stain?

Has three major goals:

1. Adheres the bacteria to the slide surface.

2. Kills most microorganisms without destroying structural features.

3. Enables many stains to better penetrate or react with the microorganisms.

How can an improperly prepared smear affect your gram stain results?

-Large purple geometric crystals or large purple "star bursts" = the slide was too warm when the crystal violet was applied causing the dye to precipitate onto the slide.

-Nothing visible on slide = not enough bacteria on slide or slide was not heat-fixed.

-Gram-negative cells stain purple or a mix of purple and pink = smears not decolorized enough or smear was too thick and ethanol was unable to decolorize the bacteria.

-Gram-positive cells stain light purple or pink = smears were heat-fixed too long, insufficient iodine used, cultures used are old, or too much water used between steps.

What is the term for the liquid used to promote primary stain retention in a gram stain?

Mordant (complexes the crystal violet and supports its retention in gram-positives).

What liquid functions as a mordant in gram-staining?

Iodine solution.

What is the primary stain in gram-staining?

Crystal violet.

What is the function of crystal violet in gram-staining?

Has a positive charge, is attracted to the negative charges associated with cell walls; stains gram-positives purple as it is complexed within the thicker peptidoglycan layer.

What is the mordant in gram-staining?

Iodine solution.

What is the function of iodine solution?

Complexes the crystal violet and supports its retention in gram-positives.

What is the decolorizer in gram-staining?

95% ethanol.

What is the function of 95% ethanol in gram-staining?

The ethanol solution is able to solubilize the dye and outer membrane associated with gram-negative cells, resulting in the removal of crystal violet dye associated with these cells.

What is the counterstain in gram-staining?

Safranin.

What is the function of safranin in gram-staining?

Enables the visualization of the gram-negative cells (stains them pink).

What type of bacteria are clustered, purple cocci?

Gram-positive.

What type of bacteria are clustered, pink rods?

Gram-negative.

If your known gram-negative rods appeared purple under the microscope, what mistake in the staining process would cause this?

Not using enough alcohol to decolorize the gram-negative cells or forgot to use it; smear is too thick.

Assume a student does a gram-stain and when they look in their microscope to see their results the slide color is very faint and almost clear, what could've been the issue?

Not enough bacteria on the slide, used too much alcohol which decolorized all cells, smears were heat-fixed too long.

Name two errors that can occur during the staining procedure and what they result in when looking at the slide through the microscope.

1. Large purple geometric crystals or large purple "star bursts" = the slide was too warm when the crystal violet was applied causing the dye to precipitate onto the slide.

2. Nothing visible on slide = not enough bacteria on slide or slide was not heat-fixed.

What are the reagents used in acid fast staining?

1. Primary stain = carbol fuchsin - contains phenol that helps solubilize the waxy cell wall and allows the stain to penetrate the cell (needs heat with it to "loosen" mycolic acid).

2. Decolorizer = acid alcohol - decolorizes all cells except acid-fast cells.

3. Counterstain = methylene blue = stains decolorized non-acid-fast cells blue.

Why do we need to use an acid-fast stain rather than a gram stain to visualize acid-fast positive cells?

Acid-fast bacteria are taxonomically gram-positive but do not stain well using that technique due to the waxy mycolic acid in their cell walls.

With gram-staining, the primary stain is dependent on reacting with the peptidoglycan layer of the cell, what part of the cell (if present) does the primary stain of acid-fast staining react with?

Mycolic acid (waxy).

What differences occur in the process of acid-fast staining vs. gram staining?

-Gram-staining = uses a mordant to lock in crystal violet.

-Acid-fast = heat must be used to lock in carbol fuchsin (steam), also dye must be prevented from drying out, no mordant.

What are the steps in focusing a microscope?

1. Turn on power and turn up light.

2. Place slide on stage, focus at 4X using coarse adjustment - wait until something is visible - then focus using fine adjustment.

3. Focus under 10X, then under 40X.

4. Place oil drop on slide, focus under 100X.

*At each step, use iris diaphragm to adjust amount of light.

What color cells are acid-fast?

Pink cells.

What are the positions of the 40x and 100x objective when adding oil to the slide?

Rotate the objective lens part way between the 40X and 100X lens.

What would the CFU/mL be for a plate with 281 colonies and a total dilution of 10^-7?

281 x 1/10^-7 = 2.81 x 10^9 CFU/mL.

Calculate the CFU for the following plate:

-Number of colonies = 150

-Dilution factor = 10^-3

-Volume plated = 0.1 mL

150 x 1/10^-3 x 1/10^-1 = 1.50 x 10^6 CFU/mL.

If a tube has a dilution of 10^-5 and you plate 0.1 mL, what is the total dilution?

10^-6.

What is the dilution in the tube if when 0.1 mL is plated, the total dilution is 10^-7?

10^-6.

What is the difference between direct and indirect quantitation?

-Direct methods employ techniques that can assess the number of individual cells or colony-forming units present in a sample (ex. microscope, cell sorter/counter, viable plate counts).

-Indirect methods evaluate a measurable metric that correlates with population size (ex. sample turbidity or measuring the mass of bacteria).

How would results be affected by an uneven distribution of bacteria over plates during the direct method of quantification of microorganisms?

There would be one large spot of bacteria, which would cause there to be an inaccurate number of CFUs and you would not be able to quantify the amount of bacteria in the original sample effectively.

What factors can lead to a slowdown in bacterial growth despite initial exponential growth?

Lack of nutrients, not enough space, buildup of waste, not enough oxygen.